Protein Protocols Protein Protocols

Ethoxyformylation of Histidine
65
Ethoxyformylation of Histidine
Dan S. Tawfik
1. Introduction
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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Diethylpyrocarbonate (DEP) reacts with various nucleophiles (amines, alcohols,
thiols, imidazoles, or guanido groups) to yield the respective ethoxyformyl derivatives.
At low pH (generally < 6.0) the reaction is quite selective for histidine, as the main side
reaction with the ε-amino group of lysine proceeds very slowly (see e.g., ref. 1). Still,
side reactions even with hydroxyl groups (e.g., of serine or tyrosine) were observed
(2). The fact that the ethoxyformyl group can be removed from the imidazole side
chain by mild treatment with hydroxylamine can be exploited to ascribe the modification
to a histidine residue. In addition, ethoxyformylation of histidines is characterized by
an increase in absorbance at 242 nm, which is also used to determine the number of
modified histidines (1–3).
2. Materials
1. (DEP) (see Note 1).
2. Acetonitrile.
3. Protein for modification (approx 5 µM) diluted in 0.1 M sodium acetate buffer, pH 5.0.
4. 1 M Hydroxylamine, pH 7.0.
3. Method
1. Prepare a series of fresh DEP solutions in acetonitrile (1–30 mM) (see Note 2).
2. Add 5-µL aliquots of each of the DEP solutions to 95-µL aliquots of the protein solution
(see Note 3).
3. Incubate for 15–60 min.
4. Determine the activity of the modified protein.
5. To assay the recovery of the ethoxyformylated histidine residues (see also Notes 4 and 5):
a. Add to a solution of the modified protein 1/10 of a volume of 1 M hydroxylamine, pH
7.0, and incubate for 10 min.
b. Dilute with acetate buffer and dialyze extensively against a buffer suitable for the
protein being studied.
c. Determine the activity of the protein.